A novel single-cell staining procedure performed in vivo under electrophysiological control: Morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin

We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: approximate to 1 mu m), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and 'tickled' by the juxtamembranous iontophoretic pulses, Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate (> 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.